Neisseria meningitidis serogroup B glycoconjugates and methods of using the same

ABSTRACT

The present invention pertains generally to novel  Neisseria meningitidis  serogroup B glycoconjugates. More particularly, the invention pertains to glycoconjugates formed from a  Neisseria meningitidis  serogroup B capsular oligosaccharide derivative (MenB OS derivative) in which sialic acid residue N-acetyl groups are replaced with N-acyl groups. The invention also pertains to vaccine formulations containing the glycoconjugates, methods of making the vaccine formulations, and methods of using the vaccine formulations to treat or prevent  Neisseria meningitidis  serogroup B or  E. coli  K1 disease in a mammalian subject.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is related to provisional patent applicationserial No. 60/024,454, filed Aug. 27, 1996, from which priority isclaimed under 35 USC §119(e)(1) and which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Technical Field

[0003] The present invention pertains generally to novel Neisseriameningitidis serogroup B glycoconjugates. More particularly, theinvention pertains to glycoconjugates formed from a Neisseriameningitidis serogroup B capsular oligosaccharide derivative (MenB OSderivative) in which sialic acid residue N-acetyl groups have beenreplace with N-acyl groups, and methods of making and using thoseglycoconjugates.

[0004] 2. Background of the Invention

[0005]Neisseria meningitidis is a causative agent of bacterialmeningitis and sepsis. Meningococci are divided into serological groupsbased on the immunological characteristics of capsular and cell wallantigens. Currently recognized serogroups include A, B, C, D, W-135, X,Y, Z and 29E. The polysaccharides responsible for the serogroupspecificity have been purified from several of these groups, includingA, B, C, D, W-135 and Y.

[0006]N. meningitidis serogroup B (“MenB”) accounts for approximately 50percent of bacterial meningitis in infants and children residing in theU.S. and Europe. The organism also causes fatal sepsis in young adults.In adolescents, experimental MenB vaccines consisting of outer membraneprotein (OMP) vesicles have been found to be approximately 50%protective. However, no protection has been observed in vaccinatedinfants and children, the age groups at greatest risk of disease.Additionally, OMP vaccines are serotype- and subtype-specific, and thedominant MenB strains are subject to both geographic and temporalvariation, limiting the usefulness of such vaccines.

[0007] Effective capsular polysaccharide-based vaccines have beendeveloped against meningococcal disease caused by serogroups A, C, Y andW135. However, similar attempts to develop a MenB polysaccharide vaccinehave failed due to the poor immunogenicity of the capsular MenBpolysaccharide (termed “MenB PS” herein). MenB PS is a homopolymer of(N-acetyl (α2→8) neuraminic acid. Escherichia coli K1 has the identicalcapsular polysaccharide. Antibodies elicited by MenB PS cross-react withhost polysialic acid (PSA). PSA is abundantly expressed in fetal andnewborn tissue, especially on neural cell adhesion molecules (“NCAMs”)found in brain tissue. PSA is also found to a lesser extent in adulttissues including in kidney, heart and the olfactory nerve. Thus, mostanti-MenB PS antibodies are also autoantibodies. Such antibodiestherefore have the potential to adversely affect fetal development, orto lead to autoimmune disease.

[0008] MenB PS derivatives have been prepared in an attempt tocircumvent the poor immunogenicity of MenB PS. For example, C₄-C₈N-acyl-substituted MenB PS derivatives have been described. See, EPPublication No. 504,202 B, to Jennings et al. Similarly, U.S. Pat. No.4,727,136 to Jennings et al. describes an N-propionylated MenB PSmolecule, termed “NPr-MenB PS” herein. Mice immunized with NPr-MenB PSglycoconjugates were reported to elicit high titers of IgG antibodies.Jennings et al. (1986) J. Immunol. 137:1708. In rabbits, two distinctpopulations of antibodies, purportedly associated with two differentepitopes, one shared by native MenB PS and one unshared, were producedusing the derivative. Bactericidal activity was found in the antibodypopulation that did not cross react with MenB PS. Jennings et al. (1987)J. Exp. Med. 165:1207. The identity of the bacterial surface epitope(s)reacting with the protective antibodies elicited by this conjugateremains unknown.

[0009] Although the above-described MenB PS derivatives are capable ofeliciting a significant anti-MenB PS response, responding antibodiesstill include a significant proportion of molecules that arecross-reactive with polysialic acid residues in host tissue, andtherefore autoreactive. Thus, to date, no approach which has been takenwith respect to MenB vaccine development has been succesful in providinga safe and effective vaccine against MenB. Accordingly, there remains aneed to provide MenB immunogens which can be used in vaccineformulations, wherein the immunogens do not elicit the production ofantibodies in immunized animals that are cross-reactive with host tissueand can be thus used in the prevention or treatment of MenB disease.

SUMMARY OF THE INVENTION

[0010] The present invention is based on the discovery that asubstantially homogenous preparation of MenB oligosaccharide (MenB OS)derivative fragments, and glycoconjugates made from those fragments,provide highly effective immunogens for use in anti-MenB vaccinepreparations. Antibodies elicited in immunized animals by these MenB OSderivative fragments do not substantially cross-react with host tissueas determined using several binding assays described herein, and aretherefore not autoreactive. Since the present MenB OS fragments do notelicit the formation of autoreactive molecules, they provide a safe andefficacious vaccine component for use in the prevention of MenB and E.coli K1 disease.

[0011] Accordingly, in one embodiment, the subject invention is directedto a glycoconjugate comprising a MenB OS derivative having sialic acidresidue N-acetyl groups replaced with N-acyl groups, wherein the MenB OSderivative is covalently attached to a carrier molecule and has anaverage degree of polymerization (Dp) of about 10 to about 20.

[0012] In another embodiment, the subject invention is directed to aglycoconjugate comprising a MenB OS derivative having sialic acidresidue N-acetyl groups replaced with N-propionyl groups, wherein theMenB OS derivative is covalently attached to a tetanus toxoid proteincarrier and has an average Dp of about 12 to about 18.

[0013] In yet another embodiment, the invention is directed to a methodfor producing a glycoconjugate comprising:

[0014] (a) providing a heterogenous population of MenB OS derivativeswherein sialic acid residue N-acetyl groups have been replaced withN-acyl groups;

[0015] (b) obtaining a substantially homogenous group of MenB OSderivatives from the population of (a) wherein the MenB OS derivativeshave an average Dp of about 10 to 20;

[0016] (c) introducing a reactive group at a nonreducing end of thederivatives obtained in step (b) to provide single end-activated MenB OSderivatives; and

[0017] (d) covalently attaching the end-activated MenB OS derivatives toa carrier molecule to provide a MenB OS glycoconjugate comprisingsubstantially homogenous sized MenB OS moieties.

[0018] In still a further embodiment, the invention is directed to amethod for producing a glycoconjugate comprising:

[0019] (a) providing a heterogenous population of MenB OS derivativeswherein sialic acid residue N-acetyl groups have been replaced withN-propionyl groups;

[0020] (b) obtaining a substantially homogenous group of MenB OSderivatives from the population of (a) wherein the MenB OS derivativeshave an average Dp of about 12 to 18;

[0021] (c) introducing a reactive group at a nonreducing end of thederivatives obtained in step (b) to provide single end-activated MenB OSderivatives; and

[0022] (d) covalently attaching the end-activated MenB OS derivatives toa tetanus toxoid carrier molecule to provide a MenB OS/tetanus toxoidglycoconjugate comprising substantially homogenous sized MenB OSmoieties.

[0023] In still further embodiments, the subject invention relates toglycoconjugates produced by these methods, to vaccine compositionscomprising the glycoconjugates in combination with a pharmaceuticallyacceptable excipient, and to methods of forming the vaccinecompositions.

[0024] In another embodiment, the subject invention is directed to amethod for preventing or treating MenB and/or E. coli K1 disease in amammalian subject comprising administering a therapeutically effectiveamount of the above vaccine compositions to the subject.

[0025] These and other embodiments of the present invention will readilyoccur to those of ordinary skill in the art in view of the disclosureherein.

BRIEF DESCRIPTION OF THE FIGURES

[0026]FIG. 1 depicts the CONJ-1 NPr-MenB OS derivative-basedglycoconjugate produced in the practice of the invention.

[0027]FIG. 2 depicts the CONJ-2 NPr-MenB OS derivative-basedglycoconjugate produced in the practice of the invention.

[0028]FIG. 3 depicts the CONJ-3 NPr-MenB OS derivative-basedglycoconjugate produced in the practice of the invention.

[0029]FIG. 4 depicts the CONJ-4 NPr-MenB OS derivative-basedglycoconjugate produced in the practice of the invention.

[0030]FIG. 5 depicts chromatograms taken during preparation of a controlNPr-MenB PS//CRM₁₉₇ glycoconjugate before and after covalent attachmentof the saccharides to the protein carrier as described in Example 2.

[0031]FIG. 6 depicts the results of the ELISA described in Example 3evaluating the production of an IgG anti-NPr-MenB PS antibody responsein animals immunized with a vaccine composition containing the controlglycoconjugates.

[0032]FIG. 7 depicts the results of the ELISA described in Example 4evaluating the IgG subclass of the antibody response elicited by acontrol glycoconjugate vaccine composition.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The practice of the present invention will employ, unlessotherwise indicated, conventional methods of immunology, microbiology,molecular biology and recombinant DNA techniques within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,Sambrook, et al. Molecular Cloning: A Laboratory Manual (2nd Edition,1989); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., 1985); Transcription andTranslation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R.Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning(1984); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weirand C. C. Blackwell eds., 1986, Blackwell Scientific Publications).

[0034] All publications, patents and patent applications cited herein,whether supra or infra, are hereby incorporated by reference in theirentirety.

[0035] As used in this specification and the appended claims, thesingular forms “a,” “an” and “the” include plural references unless thecontent clearly dictates otherwise.

I. Definitions

[0036] In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

[0037] As used herein, a “MenB PS derivative” refers to a moleculeobtained by the chemical modification of the native capsularpolysaccharide of MenB. Such MenB PS derivatives include, but are notlimited to, MenB PS molecules which have been modified by thesubstitution of sialic acid residue N-acetyl groups of the nativemolecule with appropriate acyl groups, such as C3-C₈, and higher, acylgroups wherein the term “acyl group” encompasses any acylated linear,branched, aliphatic or aromatic molecule. A particularly preferred MenBPS derivative for use herein comprises the substitution of N-propionylgroups for N-acetyl groups of native MenB PS (termed “NPr-MenB PS”herein). Methods for synthesizing N-acyl-substituted MenB PSderivatives, including NPr-MenB PS, are known in the art and describedin e.g., U.S. Pat. No. 4,727,136 to Jennings et al. and EP PublicationNo. 504,202 B, also to Jennings et al.

[0038] An “antigen” is defined herein to include any substance that maybe specifically bound by an antibody molecule. An “immunogen” is anantigen that is capable of initiating lymphocyte activation resulting inan antigen-specific immune response. Such activation generally resultsin the development of a secretory, cellular and/or antibody-mediatedimmune response against the immunogen. Usually, such a response includesbut is not limited to one or more of the following effects; theproduction of antibodies from any of the immunological classes, such asIgA, IgD, IgE, IgG or IgM; the proliferation of B and T lymphocytes; theprovision of activation, growth and differentiation signals toimmunological cells; expansion of helper T cell, suppressor T cell,and/or cytotoxic T cell and/or γδ T cell populations. Immunogenstherefore include any molecule which contain one or more antigenicdeterminants (e.g., epitopes) that will stimulate a host's immune systemto initiate such an antigen-specific response.

[0039] By “epitopel” is meant a site on an antigen to which specific Bcells and T cells respond. The term is also used interchangeably with“antigenic determinant” or “antigenic determinant site.” A peptideepitope can comprise 3 or more amino acids in a spatial conformationunique to the epitope. Generally, an epitope consists of at least 5 suchamino acids and, more usually, consists of at least 8-10 such aminoacids. Methods of determining spatial conformation of amino acids areknown in the art and include, for example, x-ray crystallography and2-dimensional nuclear magnetic resonance. Furthermore, theidentification of epitopes in a given protein is readily accomplishedusing techniques well known in the art. See, e.g., Geysen et al. (1984)Proc. Natl. Acad. Sci. USA 81:3998 (general method of rapidlysynthesizing peptides to determine the location of immunogenic epitopesin a given antigen); U.S. Pat. No. 4,708,871 (procedures for identifyingand chemically synthesizing epitopes of antigens); and Geysen et al.(1986) Molecular Immunology 23:709-715 (technique for identifyingpeptides with high affinity for a given antibody). Antibodies thatrecognize the same epitope can be identified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen.

[0040] A “unique MenB epitope” is defined herein as an epitope presenton a MenB bacterium, wherein antibodies directed toward the epitope arecapable of binding specifically to MenB and not cross reacting, orminimally cross reacting, with sialic acid residues present on thesurface of host tissue. Immunogens containing one or more “unique MenBepitopes” are thus useful in vaccines for the prevention of MenBdisease, and either will not elicit an autoimmune response, or poseminimal risk of eliciting an autoimmune response.

[0041] By “mammalian subject” is meant any member of the class Mammalia,including, without limitation, humans and other primates, including suchnon-human primates as chimpanzees and other apes and monkey species;farm animals such as cattle, sheep, pigs, goats and horses; domesticmammals such as dogs and cats; and laboratory animals including rodentssuch as mice, rats and guinea pigs. The term does not denote aparticular age or sex. Thus, both adult and newborn individuals, as wellas fetuses, either male or female, are intended to be covered.

II. Modes of Carrying Out the Invention

[0042] As explained above, the native capsular polysaccharide of MenB,termed “MenB PS” herein, is poorly immunogenic in humans andexperimental animals. Furthermore, native MenB PS elicits the productionof autoantibodies and, hence, may be inappropriate for use in vaccinecompositions. Thus, the present invention uses MenB OS derivativeimmunogens that have been selected based on their ability to elicit theformation of antibodies exhibiting functional activity against MenBbacteria, wherein such functional activity is important in conferringprotection against MenB disease. The immunogens are also selected on thebasis of eliciting an immune response that has minimal or substantiallyundetectable autoimmune activity as determined using the assaysdescribed herein.

[0043] More particularly, MenB PS derivatives were prepared for use asstarting material in the production of MenB OS derivative immunogens.The MenB PS derivatives generally comprise C₃-C₈ acyl substitutions ofsialic acid residue N-acetyl groups of the native molecule. Particularlypreferred MenB PS derivatives comprise the substitution of N-propionylgroups for N-acetyl groups of native MenB PS and are termed “NPr-MenBPS” herein. Such derivatives and methods for synthesizing the same aredescribed in e.g., U.S. Pat. No. 4,727,136 and EP Publication No.504,202 B, both to Jennings et al.

[0044] The C₃-C₈ acyl derivatives can be made by first N-deacylatingnative MenB (obtained from e.g., N. meningitidis cultures) in thepresence of a strong base to quantitatively remove the N-acetyl groupsand to provide a reactive amine group in the sialic acid residue partsof the molecule. The deacylated MenB polysaccharides are thenN-acylated. For example, in the case of NPr-MenB PS, the deacylatedmolecule is N-propionylated using a source of propionyl groups such aspropionic anhydride or propionyl chloride, as described in U.S. Pat. No.4,727,136 to Jennings et al. The extent of N-acylation can be determinedusing, for example, NMR spectroscopy. In general, reaction conditionsare selected such that the extent of N-acylation is at least about 80%.

[0045] A heterogenous population of high molecular weight MenB PSderivative molecules is thus obtained. Previous methods have used suchheterogenous high molecular weight derivatives in glycoconjugatepreparations, where the derivatives are subjected to controlledperiodate oxidation to create terminal aldehyde group at thenon-reducing end for conjugation (through the aldehydric group at thenon-reducing end of the polysaccharide) to a protein carrier. However,in the practice of the present invention, the above-described N-acylatedMenB polysaccharide derivatives are fragmented and thensize-fractionated to provide a substantially homogeneous population ofintermediate “sized” MenB oligosaccharide fragments for use in preparingglycoconjugates.

[0046] In order to provide N-acylated MenB OS derivative-basedglycoconjugates having well defined and controlled structuralconfigurations, intermediate sized N-acylated MenB oligosaccharides areprepared to have substantially homogenous saccharide moiety sizes.Glycoconjugates formed from these sized molecules are expected toexhibit more consistent immunological behavior than heterogenouspreparations. Specifically, an N-acylated MenB PS preparation, havingsubstantially 100% N-acylated sialic acid residues, as determined by,e.g., NMR analysis, can be fragmented under mild acidic conditions toprovide a population of oligosaccharide molecules of varying sizes. Thefragmented products are size fractionated, using for example, standardchromatographic techniques combined with e.g., stepwise salt gradients,to provide fractions of N-acylated MenB molecules of homogenous sizes.Fractions containing intermediate sized oligosaccharides e.g., with anaverage Dp of about 5 to about 25, preferably 10 to about 20, and moreparticularly about 12 to about 18, are selected for further use herein.These sized N-acylated oligomers having intermediate length are smallenough to function as a T-cell-dependent hapten, yet are large enough toexpress relevant conformational epitopes.

[0047] In order to increase the immunogenicity of the sized MenB PSderivative fragments, the molecules can be conjugated to a suitablecarrier molecule to provide glycoconjugates. Suitable carriers aredescribed herein further below. Particularly, glycoconjugatepreparations having well defined and controlled structuralconfigurations can be formed from intermediate sized N-acylated MenBoligosaccharides to provide N-acylated MenB oligosaccharide immunogenshaving superior immunogenicity.

[0048] Thus, in one embodiment of the invention, a group of N-acylatedMenB OS glycoconjugates, an example of which is termed “CONJ-1” herein,can be prepared as follows. Fractions of intermediate sizedoligosaccharides having an average Dp of about 10 to about 20, andpreferably about 12 to about 18, are chemically end-activated at theirnon-reducing termini and conjugated to protein carriers by a reductiveamination technique to provide the CONJ-1 glycoconjugates. The resultingglycoconjugate is depicted in FIG. 1, wherein the oligosaccharidefragments 2 are shown covalently linked at their nonreducing ends 4 to asuitable protein carrier 6 to provide a glycoconjugate, generallyindicated at 8. Successful conjugation can be determined using, forexample, gel filtration, and the final saccharide to protein ratio (w/w)assessed by calorimetric assay.

[0049] In a related embodiment of the invention, another group ofN-acylated MenB OS glycoconjugates, an example of which is termed“CONJ-2” herein, can be prepared as follows. Fractions of intermediatesized oligosaccharides having an average Dp of about 10 to about 20, andpreferably about 12 to about 18, are anchored to a protein carrier attheir reducing ends to provide glycoconjugtes having a reversed chemicalpolarity (orientation). In particular, the reducing ends of theN-acylated MenB oligosaccharide fragments can be converted to free aminogroups by reductive amination using, for example, NaCNBH₃. The freeamino groups can then be modified by covalently attaching an anchoringmolecule bearing an N—OH succinimide active ester of adipic acid.Conjugation to a protein carrier occurs by nucleophilic displacement ofthe active ester group with the ε-amino group of lysine to provide astable amide bond. As can be seen by reference to FIG. 2, the resultingCONJ-2 glycoconjugates, generally indicated at 18, have a configurationsimilar to the CONJ-1 glycoconjugates, however, the saccharide fragmentsare oriented in the opposite direction relative to the protein carrier.This structural orientation more closely resembles the native chemicalpolarity of MenB PS. In particular, the oligosaccharide fragments 12 areshown covalently linked at their reducing ends 15 to a suitable proteincarrier 16 to provide the CONJ-2 glycoconjugate 18.

[0050] In order to provide CONJ-2 glycoconjugates wherein theoligosaccharide fragments are projected away from the protein carrier,the above method can be altered by using a hydrocarbon spacer armbearing the N—OH succinimide active ester of adipic acid to modify thefree amino group on the aminated molecules. The spacer arm can include aC3-C8 molecule which extends the oligosaccharide fragments away from theprotein carrier in the glycoconjugate.

[0051] Yet further glycoconjugates can be formed from theabove-described sized MenB OS derivative fragments. In particular, thepresence of a lipid moiety at the reducing ends of bacterial MenB PS hasbeen demonstrated. Mandrell et al. (1982) J. Immunol. 129:2172. Notbeing bound by any particular theory, this lipid moiety may act as ananchoring mechanism, binding the polysaccharide to the bacterial surfacethrough hydrophobic interactions. Thus, the saccharide-lipid junctionalarea of native MenB PS may provide unique epitopes (neo-determinants)that are not presented in purified MenB PS preparations, either as aresult of masking and/or shielding effects of the architecture of thelong polysialic acid chain, or due to the loss of the lipid moietyduring purification.

[0052] Accordingly, in still further related embodiments of theinvention, MenB OS glycoconjugates are provided, examples of which aretermed “CONJ-3” and “CONJ-4” herein, wherein the glycoconjugates areconstructed to have enhanced physicochemical and immunologicalproperties due to the addition of lipid moieties which provide a mimicof the native MenB saccharide-lipid junctional area. In one particularembodiment, a substantially homogeneous fraction of intermediate sizedoligosaccharides having an average Dp of about 15 to about 25 can beobtained as described above. These oligosaccharide fragments should havesufficient length to fold into important conformational epitopes (e.g.,extended helixes), yet are not too long to exert substantial stericmasking or shielding of potential saccharide-lipid junctionneo-epitopes. Hydrocarbon chains of varying length, for example C3-C16long-chain aliphatic lipids, such as phosphatidylethanolamine or otherlipid molecules containing propionyl, hexanoyl and dodecanoyl groups,can be covalently attached at the reducing end of the MenB OS fragmentsusing the above-described N—OH active ester coupling procedure. Theresulting alkylated sialo-oligomers can then be subjected to mildcontrolled periodate oxidation to introduce terminal free aldehydegroups at the nonreducing ends of the oligosaccharide moieties. Thesemonovalent alkylated-sialo-oligomers can then be coupled to a suitableprotein carrier by reductive amination to provide CONJ-3glycoconjugates. Referring to FIG. 3, a CONJ-3 glycoconjugate isgenerally indicated at 28. The glycoconjugate comprises lipid moieties32 covalently attached at the reducing ends 25 of intermediate sizedMenB OS derivative fragments 22 to provide monovalentalkylated-sialo-oligomers, generally indicated at 34. Thealkylated-sialo-oligomers 34 are coupled to a protein carrier 26 at thenonreducing end 24 of the MenB OS fragments 22.

[0053] In a related embodiment, a substantially homogeneous fraction ofintermediate sized oligosaccharides having an average Dp of about 15 toabout 25 can be covalently coupled to C3-C16 aliphatic lipids at thenonreducing termini of the MenB OS derivative fragments using periodateoxidation and selective reductive techniques. The resultingalkylated-sialo-oligomers can then be coupled to a suitable proteincarrier by first converting the reducing ends of the MenBoligosaccharide derivative moieties to free amino groups by reductiveamination. The free amino groups can then be modified by covalentlyattaching an anchoring molecule bearing an N—OH succinimide active esterof adipic acid. Optionally, a C3-C8 spacer arm bearing the active estergroup can be added to project the alkylated-sialo-oligomers away fromthe protein carrier. Conjugation to a protein carrier occurs bynucleophilic displacement of the active ester group with the ε-aminogroup of lysine to provide a stable amide bond to provide CONJ-4glycoconjugates. Referring to FIG. 4, a CONJ-4 glycoconjugate isgenerally indicated at 58. The glycoconjugate includes lipid moieties 62covalently attached at the nonreducing ends 54 of intermediate sizedMenB OS derivative fragments 52 to provide monovalentalkylated-sialo-oligomers, generally indicated at 64. Thealkylated-sialo-oligomers 64 are coupled to a protein carrier 56 at thereducing end 55 of the MenB OS fragments 52.

[0054] In both of the CONJ-3 and CONJ-4 glycoconjugates, thelipid-saccharide junctional region is exposed by being arranged distalto the protein carrier. This configuration renders the lipid-saccharidejunctional region (neo-epitopes) immunologically accessible andrecognizable for inducing antibody formation to the neo-epitope regions.The CONJ-4 glycoconjugates have a similar structure to the CONJ-3glycoconjugates, however, the saccharide fragments are oriented in theopposite direction relative to the protein carrier.

[0055] In addition to providing glycoconjugates such as CONJ-3 andCONJ-4 which have artificially generated MenB OS derivatives withlipidated ends, native MenB oligomers which contain thenaturally-occurring lipid are isolated and purified for use in preparingglycoconjugate preparations. Thus in another embodiment of theinvention, MenB PS can be digested using neuraminidase (rather than acidhydrolysis as described above) which preserves the structural andchemical integrity of the saccharide-lipid portion of the native MenB PSchain. In this manner, sialic acid residues are sequentially removedfrom the nonreducing terminus by the action of the neuraminidase enzyme.By using time-controlled digestion, substantially homogenous fractionsof sialyl-lipid oligomers can be generated having varying chain lengths.The resultant free sialic acid residues can be removed from thepreparation by dialysis, and the retentate, containing the lipid-MenBoligomers can be purified by ion-exchange or hydrophobic interactionchromatography techniques. The lipid-MenB oligomers are then availablefor conjugation to suitable protein carriers using the techniquesdescribed above. In particular, conjugation will generally involveselective end-group activation at the nonreducing end of the lipid-menBoligomers to allow single-site covalent attachment to the carriermolecules.

[0056] Each of the above-described glycoconjugates are prepared usingcarrier molecules that will not themselves induce the production ofharmful antibodies. Suitable carriers are typically large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,lipid aggregates (such as oil droplets or liposomes), and inactive virusparticles. Preferably, the sized MenB OS derivative fragments of thepresent invention are conjugated to a bacterial toxoid, such as but notlimited to a toxoid from diphtheria, tetanus, cholera, etc. Inparticular embodiments, the oligosaccharide fragments are coupled to theCRM₁₉₇ protein carrier. The CRM₁₉₇ carrier is a well-characterizednon-toxic diphtheria toxin mutant that is useful in glycoconjugatevaccine preparations intended for human use. Bixler et al. (1989) Adv.Exp. Med. Biol. 251:175, Constantino et al. (1992) Vaccine. In otherembodiments, the MenB OS derivative fragments are coupled to proteincarriers known to have potent T-cell epitopes. Exemplary carriersinclude, but are not limited to, Fragment C of tetanus toxin (TT), andthe Class 1 or Class 2/3 OMPs of N. meningitidis. Such carriers are wellknown to those of ordinary skill in the art. Glycoconjugates areselected for their ability to express saccharide-associated epitopesthat mimic those found on the surface of MenB bacterial cells. Suitableglycoconjugates for use with the present invention elicit the formationof functional, bacteria-specific antibodies in immunized hosts, and donot cross-react with host tissue as determined using the binding assaysdescribed herein.

[0057] Several factors will have an impact on the physical andimmunological properties of the above-described glycoconjugates.Specifically, average MenB oligomer fragment size, ratio of saccharideto protein (hapten loading density), linkage chemistry, and the choiceof protein carrier are all factors that should be considered andoptimized in the preparation of the present glycoconjugates. Forexample, a low saccharide loading density may result in pooranti-saccharide antibody response. On the other hand, a heavy loading ofsaccharides could potentially mask important T-cell epitopes of theprotein molecule, thus abrogating the carrier effect and attenuating thetotal anti-saccharide immune response.

[0058] Accordingly, during the course of the various conjugationreactions, aliquots can be withdrawn and analyzed by SEC-HPLC in orderto monitor the extent of the conjugation process. The use of adisaggregating buffer, for example EDTA, SDS, deoxycholate, or the like,can be employed to separate components possibly adhering to thepreparations by non-covalent interactions. To ensure glycosylation ofthe carrier, the shift in retention time of the particular proteincarrier toward the exclusion volume (V₀) of the column can be monitored.In addition, a gradual reduction of the saccharide peak area in a HPLCchromatogram can be used to indicate incorporation of the saccharideonto the carrier.

[0059] Characterization of the glycoconjugates can include molecularweight determination using, for example, gel filtration columns. Furthercharacterization may also include electrophoretic mobility on SDS-PAGEseparation equipment and analysis of chemical composition of theglycoconjugates with respect to carbohydrate and amino acid components.The identity of product purity, and the absence of residual contaminants(such as nucleic acids, LPS, and free saccharides and/or carrier) canalso be verified using known techniques. Confirmation of stable covalentattachment can be accomplished using a combination of analyticaltechniques, including gel filtration in detergent-containing buffer,SDS-PAGE followed by Western Blot analysis and amino acid analysis. See,e.g., Vella et al. (1992) Vaccines: New Approaches to ImmunologicalProblems, (Ellis, R. W. ed), Butterworth-Heinemann, Boston, pp 1-22,Seid et al. (1989) Glycoconjugate J. 6:489.

[0060] The glycoconjugates of the present invention are used to elicitthe formation of an anti-MenB immune response in an immunized host.Anti-MenB antibodies produced by the immunized host should bind to MenBbacteria while not cross-reacting, or minimally cross-reacting, withhost tissue sialic acid residues as determined using the binding assaysdescribed herein. The anti-MenB antibodies can be fully characterizedwith respect to isotype, fine antigenic specificity, functional activityand cross-reactivity with polysialic acid residues in host tissue.Glycoconjugates capable of eliciting non-autoreactive, IgG antibodieshaving bactericidal activity are selected for use in preparing vaccineformulations for use in anti-MenB immunization.

[0061] For example, immunogenicity of MenB OS derivative glycoconjugatescan be determined by challenging mammalian subjects, conveniently,standard laboratory animals such as rodents and rabbits, withcompositions containing the glycoconjugates along with a suitableadjuvant, described further below. Groups of subjects are generallyimmunized and boosted several times with the compositions, or withcontrol materials (e.g., adjuvant alone, native MenB PS, MenB OSderivative fragments, or non-covalent MenB OS derivative/carriercomplexes). Antisera from immunized subjects can be obtained, and serialdilutions of pooled sera evaluated by, e.g., ELISA using standardtechniques. Labeled anti-IgG sera can be used to measure IgG anti-MenBOS derivative antibody response. In order to determine the isotypes ofthe antibodies elicited by the conjugates, standard methods, such asELISAs, can also be run using labelled molecules specific for IgGsubclasses IgG1, IgG2a, IgG2b and IgG3. An isotypic response that ispredominantly IgG1 along with IgG2b and, to a lesser extent, IgG2a andIgG3 is characteristic of a T-cell dependent antigen. Conjugates thatare found to be highly immunogenic and produce predominantly IgGantibodies are selected for further evaluation.

[0062] In particular, the specificity of the antibodies elicited byselected MenB OS derivative glycoconjugates can be further evaluatedusing competitive specific binding assays, such as inhibition ELISA, orthe like. For example, antisera obtained from immunized subjects, alongwith either soluble MenB OS derivatives (or glycoconjugates) or nativeMenB PS, can be reacted with bound MenB OS derivatives (orglycoconjugates thereof) in a suitable ELISA reaction vessel usinglabeled anti-Ig (anti-IgM, IgG and IgA) as the secondary antibody. MenBOS glycoconjugates that elicit the formation of antibodies that areinhibited to a greater extent by the soluble MenB OS derivatives andglycoconjugates than by the soluble native MenB PS (e.g., that elicitantibodies which exhibit a higher affinity for the modifiedpolysaccharide molecule) are thus selected as candidates for use infurther immunization studies.

[0063] Functional activity can be determined by assessingcomplement-mediated bactericidal activity and/or opsonic activity. Inparticular, complement-mediated bactericidal activity of the antibodiescan be evaluated using standard assays such as those described by Goldet al. (1970) Infect. Immun. 1:479, Westerink et al. (1988) Infect.Immun. 56:1120, Mandrell et al. (1995) J. Infect. Dis. 172:1279, andGranoff et al. (1995) Clin. Diagn. Laboratory Immunol. 2:574. In theseassays, N. meningitidis is reacted with a complement source as well aswith the antibody to be tested. Bacterial counts are done at varioussampling times. Those antibodies that demonstrate complement-mediatedbactericidal activity, as demonstrated by a minimum of a 50% reductionin viable bacterial cell counts determined after sixty minutesincubation with antibody and complement, as compared to colony counts attime zero, are considered to exhibit bactericidal activity for purposesof the present invention and are suitable for further use.

[0064] Complement-mediated bacteriolysis is thought to be the majormechanism responsible for host protection against invasive Meningococcaldisease. However, considerable evidence also supports an importantprotective role for opsonization (see, e.g., Bjerknes et al. (1995)Infect. Immun. 63:160). Accordingly, the opsonic activity of theantibodies produced herein can be evaluated as a second measure, or asan alternative measure, to assess functional activity. Results fromopsonic assays can be used to supplement bactericidal data, and to helpin the selection of appropriate glycoconjugates capable of conferringprotection.

[0065] A variety of opsonic assay methods are known in the art, and canbe used to evaluate functional activity of antibodies induced by theglycoconjugates of the present invention. Such standard assays includethose described by Sjursen et al. (1987) Acta Path. Microbiol. Immunol.Scand., Sec. C 95:283, Halstensen et al. (1989) Scand. J. Infect. Dis.21:267, Lehmann et al. (1991) APMIS 99:769, Halstensen et al. (1991)NIPH Annals 14:157, Fredlund et al. (1992) APMIS 100:449, Guttormsen etal. (1992) Infect. Immun. 60:2777, Guttormsen et al. (1993) J. Infec.Dis. 167:1314, Bjerknes et al. (1995) Infect. Immun. 63:160, Hayrinen etal. (1995) J. Infect. Dis. 171:1481, de Velasco et al. (1995) J. Infect.Dis. 172:262, and Verheul, A. F. M. (1991) “Meningococcal LPS DerivedOligosaccharide-Protein Conjugate Vaccines, Immunochemical andImmunological Aspects,” Thesis, Utrecht University, The Netherlands, pp.112-135.

[0066] Several binding assays can be used to evaluate possibleautoreactivity of antibodies induced by the glycoconjugates of thepresent invention. In particular, the induced antibodies can beevaluated for their ability to bind to host cells which expresspolysialic acid residues on their cell surfaces. Such cells representsurrogate targets for the detection of antibodies that exhibitautoimmune activity. One target comprises the human neuroblastoma cellline, CHP-134, which expresses long chain α2-8 polysialic acid (NCAM) onits cell surface, as described by Livingston et al. (1988) J. Biol.Chem. 263:9443. Other suitable targets include, but are not limited to,newborn brain cells, tissues derived from e.g., kidney, heart and theolfactory nerve, cultured saphenous vein endothelial cells, cytotoxic Tlymphocytes and natural killer (NK) cells. See, e.g., Brandon et al.(1993) Intl. J. Immunopathology and Pharmacology 6:77. Antibodymolecules obtained from immunized subjects can be added to suitable testcell populations in culture, and the potential binding of the antibodiesto the cellular targets detected and quantified directly using labeledmonoclonals, or indirectly using an appropriately labeled secondaryreagent that reacts specifically with the antibody (e.g., StaphylococcalProtein A and G and anti-murine antibody molecules). Antibodies that donot cross-react with test host tissue PSA or that display minimalreactivity are not considered autoreactive for purposes of the presentinvention. Thus, the glycoconjugates used to elicit formation of suchantibodies are appropriate for further use. In addition, some antibodiesthat show binding with test tissue, which binding is not affected bypre-treatment of the test cells with neuraminidase, may also beindicative of glycoconjugates that are appropriate for further use.Autoreactivity of such antibodies is termed “indeterminate” herein.

[0067] The processes used to provide the various MenB OS-derivativeconjugates are designed to produce superior immunogens presenting uniquesaccharide-associated epitopes that mimic those found on the surface ofMenB organisms and are expressed minimally in the host. The saccharidederivatives described herein are thus capable of eliciting theproduction of MenB-specific antibodies, and are used directly inanti-MenB vaccine formulations which can be used in pharmaceuticalcompositions to prevent and/or treat MenB and E. coli K1 disease inmammals. Such disease includes bacterial meningitis and sepsis ininfants, children and adults.

[0068] The vaccines can comprise one or more of the MenB OS derivativeimmunogens. The vaccines may also be administered in conjunction withother antigens and immunoregulatory agents, for example,immunoglobulins, cytokines, lymphokines, and chemokines, including butnot limited to IL-2, modified IL-2 (cys125→ser125), GM-CSF, IL-12,γ-interferon, IP-10, MIP1β and RANTES.

[0069] The vaccines will generally include one or more “pharmaceuticallyacceptable excipients or vehicles” such as water, saline, glycerol,ethanol, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, and the like, may bepresent in such vehicles.

[0070] Adjuvants may also be used to enhance the effectiveness of thevaccines. Adjuvants can be added directly to the vaccine compositions orcan be administered separately, either concurrent with or shortly after,vaccine administration. Such adjuvants include, but are not limited to:(1) aluminum salts (alum), such as aluminum hydroxide, aluminumphosphate, aluminum sulfate, etc.; (2) oil-in-water emulsionformulations (with or without other specific immunostimulating agentssuch as muramyl peptides (see below) or bacterial cell wall components),such as for example (a) MF59 (International Publication No. WO90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85(optionally containing various amounts of MTP-PE (see below), althoughnot required) formulated into submicron particles using a microfluidizersuch as Model 110Y microfluidizer (Microfluidics, Newton, Mass.), (b)SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymerL121, and thr-MDP (see below) either microfluidized into a submicronemulsion or vortexed to generate a larger particle size emulsion, and(c) Ribi™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.)containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cellwall components from the group consisting of monophosphorylipid A (MPL),trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferablyMPL+CWS (Detox™); (3) saponin adjuvants, such as Stimulon™ (CambridgeBioscience, Worcester, Mass.) may be used or particle generatedtherefrom such as ISCOMs (immunostimulating complexes); (4) CompleteFreunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5)cytokines, such as interleukins (IL-1, IL-2, etc.), macrophage colonystimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; and (6)other substances that act as immunostimulating agents to enhance theeffectiveness of the composition.

[0071] Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

[0072] Typically, the vaccine compositions are prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared. The preparation also may be emulsified or encapsulatedin liposomes for enhanced adjuvant effect, as discussed above.

[0073] The vaccines will comprise a therapeutically effective amount ofthe MenB OS derivative glycoconjugate immunogen, and any other of theabove-mentioned components, as needed. By “therapeutically effectiveamount” is meant an amount of a molecule which will induce animmunological response in the individual to which it is administeredwithout stimulating an autoimmune response. Such a response willgenerally result in the development in the subject of a secretory,cellular and/or antibody-mediated immune response to the vaccine.Usually, such a response includes but is not limited to one or more ofthe following effects; the production of antibodies from any of theimmunological classes, such as immunoglobulins A, D, E, G or M; theproliferation of B and T lymphocytes; the provision of activation,growth and differentiation signals to immunological cells; expansion ofhelper T cell, suppressor T cell, and/or cytotoxic T cell and/or γδ Tcell populations.

[0074] Preferably, the effective amount is sufficient to bring abouttreatment, i.e., reduction or complete elimination of symptoms, orprevention of disease symptoms. The exact amount necessary will varydepending on the subject being treated; the age and general condition ofthe subject to be treated; the capacity of the subject's immune systemto synthesize antibodies; the degree of protection desired; the severityof the condition being treated; the particular molecule selected and itsmode of administration, among other factors. An appropriate effectiveamount can be readily determined by one of skill in the art. A“therapeutically effective amount” will fall in a relatively broad rangethat can be determined through routine trials.

[0075] Once formulated, the vaccines are conventionally administeredparenterally, e.g., by injection, either subcutaneously orintramuscularly. Additional formulations suitable for other modes ofadministration include oral and pulmonary formulations, suppositories,and transdermal applications. Dosage treatment may be a single doseschedule or a multiple dose schedule.

III. Experimental

[0076] Below are examples of specific embodiments for carrying out thepresent invention. The examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

[0077] Efforts have been made to ensure accuracy with respect to numbersused (e.g., amounts, temperatures, etc.), but some experimental errorand deviation should, of course, be allowed for.

EXAMPLE 1 Preparation of Control MenB PS Conjuqates

[0078] Purified MenB PS in its sodium form was deacylated using 2M NaOHand NaBH₄ at about 110° C. for 6 hours to quantitatively remove theN-acetyl groups. The deacylated MenB PS was N-propionylated by use ofpropionic anhydride to yield NPr-MenB PS, as described in U.S. Pat. No.4,727,136 to Jennings et al. The extent of N-propionylation wasestimated to be around 84% by ¹H-NMR spectroscopy. The NPr-MenB PS waspurified by dialyzing against distilled water, and subjected to mildperiodate oxidation to introduce a terminal aldehydric group at thenon-reducing end for subsequent conjugation to a protein carrier, asdescribed in U.S. Pat. No. 4,727,136 to Jennings et al. During the mildperiodate oxidation, the NPr-MenB PS was fragmented, giving rise to aheterogenous population of “unsized” NPr-MenB oligosaccharide fragments,typically having an average DP of greater than about 30.

[0079] Conjugation of the unsized NPr-MenB PS fragments to two differentprotein carriers, TT and CRM₁₉₇, was performed by reductive amination inthe presence of sodium cyanoborohydride. The conjugation reaction wascarried out over about 5 days at 40° C. Two control MenB PS conjugateswere thus obtained, C1/TT and C1/CRM₁₉₇.

EXAMPLE 2 Characterization of the Control Conjugates

[0080] SDS-PAGE and Sephadex G-100 gel filtration were carried out inorder to confirm formation of covalent conjugate moieties. Referring toFIG. 1, the results of a typical Sephadex G-100 gel filtration of theC1/CRM₁₉₇ conjugate is depicted. In particular, the chromatogram of anoncovalent mixture of the NPr-MenB PS fragments and the CRM₁₉₇ carriermolecule prior to conjugation, (e.g., before the addition of NaCNBH₃) isdepicted in the top panel of FIG. 1. As can be seen, the NPr-MenBsaccharides eluted as a broad peak near the bed volume, while the CRM₁₉₇protein eluted slightly ahead of bed volume. The chromatogram afterconjugation (e.g., following the addition of NaCNBH₃ to effect reductiveamination) is depicted in the bottom panel of FIG. 1. As shown therein,a new high molecular weight (HMW) peak appeared near the void volume.This HMW peak, containing both saccharide and protein, was collected andidentified as the C1/CRM₁₉₇ conjugate.

[0081] The final saccharide-to-protein ratios (w/w) of the two C1conjugates were determined by calorimetric assays and found to be 0.21for the C1/CRM₁₉₇ conjugate and 0.15 for C1/TT conjugate.

EXAMPLE 3 Immunogenicity of the Control Conjugates

[0082] In order to assess the ability of the C1 control conjugates toelicit an IgG anti-NPr-MenB PS antibody response in an immunizedsubject, the following study was carried out. Groups of CD1 mice (10animals/group) were immunized three times by intraperitoneal (ip)injection using vaccine formulations containing C1/TT or C1/CRM₁₉₇conjugates with FCA or alum adjuvant (5.0 μg sialic acid content in theconjugate vaccine for the first injection, and 2.5 μg sialic acidcontent in the second and third doses). Control groups were immunizedwith either adjuvant alone; native MenB PS; NPr-MenB PS; ornoncovalently associated NPr-MenB PS/carrier complexes.

[0083] Serum samples were collected and pooled from each experimentalgroup concurrently with each immunization boost, as well as 11 daysafter the final boost. Serial dilutions of pooled sera were made andevaluated by ELISA using an avidin-biotinylated NPr-MenB PS system.After overnight incubation with the sera, the reaction wells wereincubated for 3 hours with alkaline phosphatase-labelled anti-murinesera specific to IgG. After washing, p-nitrophenyl phosphate was addedto the wells, and the optical density (“OD”) values were read at 405 nmafter 30 minutes color development. The OD values are reported in FIG.2. Both the C1/TT and C1/CRM₁₉₇ conjugates were immunogenic-whenadministered with FCA, and immunogenic to a lesser extent whenadministered with the alum adjuvant. The OD values of the pooled serafrom animals immunized with the conjugate/FCA vaccine formulations areshown at 1:1600 sera dilution, while the OD values of the pooled serafrom animals immunized with the conjugate/alum vaccine formulations areshown at 1:400 sera dilution. As can be seen by reference to FIG. 2,there was no significant difference observed in immunogenicity due tothe particular protein carrier used (TT or CRM₁₉₇); however, use of theFCA adjuvant greatly increased the immunogenicity of the vaccinecompositions.

EXAMPLE 4 Characterization of the Antibody Response Elicited by the C1Conjuqates

[0084] In order to evaluate the IgG subclass of the antibody responseinduced by the C1 control conjugate, the following study was carriedout. Groups of mice (10 animals per group) were given three doses ofvaccine compositions containing either C1/TT or C1/CRM₁₉₇ conjugate withFCA or alum adjuvant. The dosages were the same as those used in theimmunizations of Example 3 above. Serum samples were collected andpooled from each experimental group after the final immunization boost.Serial dilutions of pooled sera were made and evaluated by ELISA usingan avidin-biotinylated NPr-MenB PS system. After overnight incubation ofsera, reaction wells were incubated for 3 hours with alkalinephosphatase-labelled anti-murine sera specific to IgG subclasses IgG1,IgG2a, IgG2b and IgG3. After washing, p-nitrophenyl phosphate was addedto the wells, and the OD values were read at 405 nm after 30 minutescolor development. The OD values are depicted in FIG. 3, wherein thevalues represent the net OD after subtraction of blank values obtainedfrom wells containing only the colorimetric substrate.

[0085] As can be seen, the predominant antibody response was IgG1;however, when the conjugates were administered with the FCA adjuvant,there also were IgG2b and, to a lesser extent, IgG2a and IgG3 antibodyresponses. Thus, the antibody response elicited in the immunized mice byC1 conjugates is characteristic of a T-cell dependent antigen.

[0086] In order to evaluate total anti-Npr-MenB PS antibody responseinduced by the C1/TT and C1/CRM₁₉₇ conjugates in CD1 mice, and todetermine the specificity of the conjugate-induced antibody responses,the following study was carried out. Groups of CD1 mice (8 to 10 animalsper group) were immunized with three doses of conjugate vaccineformulations or control materials as described above in Example 3. Serumsamples were collected and pooled from each experimental group after thefinal immunization boost. In order to assess total Ig response to the C1conjugates, solid phase ELISA was carried out wherein biotinylatedNPr-MenB PS (bound to the reaction wells by avidin) was used as thecoating antigen. The labelling antibody was anti-murine IgM, IgG and IgAconjugated to alkaline phosphatase. After washing, p-nitrophenylphosphate was added to the wells, and the OD values were read at 405 nmafter 30 minutes color development. In order to assess specificity,competitive inhibition ELISA was carried out using the same coatingantigen with the addition of either soluble NPr-MenB PS or native MenBPS inhibitors at 25 μg/ml.

[0087] The results of both the determination of the level of totalantibody response and the specificity of the responding antibodies aredepicted below in Table 1. TABLE 1 % Inhibition Mouse 1/Titer NPr NAcGroup Vaccine Adjuvant (OD 0.5) Form Form 1 Cl (TT) FCA ˜7,000 97 28conjugate 2 Cl (TT) alum ˜1,200 99 14 conjugate 3 none FCA <100 — — 4none alum <100 — — 5 NPr-MenB OS + TT FCA <100 — — 6 NPr-MenB PS FCA<100 — — 7 NPr-MenB PS none <100 — — 8 MenB PS none <100 — — 9 Cl(CRM₁₉₇) FCA ˜6,000 98 36 conjugate 10 Cl (CRM₁₉₇) alum ˜175 56 ˜0conjugate 11 NPr-MenB alum <100 — — OS + CRM₁₉₇

[0088] As can be seen in Table 1, the CD1 mice that were immunized withthe C1 conjugate/FCA adjuvant formulations gave significant antibodyresponses to NPr-MenB PS. In addition, the antibody response wasspecific to NPr-MenB PS as demonstrated by almost complete inhibition(e.g., 97-99%) by the soluble NPr-MenB inhibitor as compared with thepartial inhibition (e.g., 14-36%) observed with the native MenB PSinhibitor. The percent inhibition with the soluble inhibitors isexpressed in Table 1 as a comparison with buffer controls.

[0089] In order to assess bactericidal activity, pooled sera obtainedfrom the above immunized subjects was added to cultures of N.meningitidis (MenB bacteria cultures) along with a source of complement.In this particular assay, a heterologous complement source (e.g.,juvenile rabbit serum) was used. Negative (sera) control and complementcontrol cultures were also assayed, and all sera were heat-inactivatedbefore testing. The results of the bactericidal assay are depicted belowin Table 2. TABLE 2 Mice Group Vaccine 1/BC₅₀ ¹  1 C1 (TT)/FCA ˜50  9 C1(CRM₁₉₇)/FCA ˜200  2 C1 (TT)/alum <25 10 C1 (CRM₁₉₇)/alum <25 3, 4, 5, 6Control Groups^(2,3) <25

[0090] In Table 2, bactericidal activity is expressed as theconcentration at which 50% of the MenB bacteria were killed relative tothe negative sera controls and complement controls. As can be seen, CD1mice immunized with the C1/TT and C1/CRM₁₉₇ conjugates administered withFCA produced antibodies that demonstrate significant bactericidalactivity.

EXAMPLE 5 Preparation of CONJ-1 MenB OS Derivative Glycoconjugates

[0091] A preparation of NPr-MenB OS derivative-tetanus toxoidconjugates, hereinafter referred to as CONJ-1, was prepared as follows.Purified MenB PS in its sodium form was deacylated with 2M NaOH at about110° C. for 6 hours to quantitatively remove the N-acetyl groups. Thealkali treatment was performed in the presence of NaBH₄. After alkalitreatment, the deacylated MenB PS was exhaustively dialyzed in saturatedsodium bicarbonate buffer. The dialyzed product was then treated with anexcess of propionic anhydride with stirring overnight at ambienttemperature to yield NPr-MenB PS. The NPr-MenB PS was exhaustivelydialyzed in water and recovered by lyophilization. The extent ofN-propionylation as measured by ¹H-NMR spectroscopy was found to besubstantially 100%.

[0092] The NPr-MenB PS was depolymerized (fragmented) under mild acidicconditions (e.g., 10 mM acetate, pH 5.5 at 50° C. for 2 hours) to give amixture of NPr-MenB oligosaccharides (NPr-MenB OS) of varying sizes. Thekinetics of hydrolysis of the NPr-MenB PS, and the resulting fragmentedoligosaccharide profile can be monitored by analytical FPLC monoQchromatography.

[0093] The mixture of fragmented NPr-MenB OS was size-fractionated onQ-Sepharose with a low (100 mM NaCl) and high (500 mM NaCl) stepwisesalt gradient. By analytical analysis, for example, the Svennerholmresorcinol assay for sialic acid (Svennerholm, L. (1957) Biochim.Biophys. Acta 24:604) and the Hantzsch calorimetric assay for releasedformaldehyde from the non-reducing end of NPr-MenB PS oligomers (Nash,T. (1953) Biochem. J. 55:416), the 100 mM NaCl fraction should containsmall-sized NPr-MenB OS molecules with an average Dp of 3-6 and the 500mM NaCl fraction should contain intermediate-sized NPr-MenB OS moleculeswith an average Dp of 13-20. As shown by analytical monoQ analysis overa Q-Sepharose column, the expected oligosaccharide distribution patternwas confirmed wherein the 100 mM NaCl fraction contained small oligomers(i.e., average Dp of 2.85) and the 500 mM NaCl fraction containedintermediate size oligomers with an average Dp of 13.

[0094] A group of intermediate-sized NPr-MenB OS derivatives (Dp of 13)recovered from the 500 mM NaCl fraction of Q-Sepharose were chemicallyend-activated at their non-reducing termini and conjugated to tetanustoxoid (TT) by a reductive amination method to provide CONJ-1glycoconjugates. More particularly, the Dp 13 oligosaccharides weresubjected to mild periodate oxidation (e.g., 100 mM sodium perborate for15-30 minutes in the dark at ambient temperature) to introduce aterminal aldehydric group at the non-reducing ends of theoligosaccharides. Following periodate oxidation, excess ethylene glycolwas used to quench the oxidation reaction. The oxidized,intermediate-sized NPr-MenB oligosaccharide derivatives were purified bydesalting on a Sephadex G-25 column and then lyophilized.

[0095] The reductive amination conjugation reaction was performed in thepresence of sodium cyanoborohydride for 3 to 5 days. For the conjugationreaction, the saccharide-to-protein ratio can range from 50 to 250mol/mol. To prepare the NPr-MenB OS/TT conjugates, the pool of NPr-MenBoligomers with average Dp13 was combined with suitably prepared TT at aninitial high molar ratio (200:1) of oligomer-to-protein. The reactionwas allowed to proceed for 3 days (e.g., 1 day at 40° C., followed by 2days at ambient temperature).

[0096] Isolation and purification of the CONJ-1 glycoconjugates can beaccomplished by gel permeation chromatography with an appropriate sizingcolumn or by hydrophobic interaction chromatography (e.g., using PhenylSepharose). Either chromatographic procedure is efficient in separatingthe glycoconjugates from reagents, byproducts, and unreacted saccharideand protein carrier molecules.

EXAMPLE 6 Characterization of the NPr-MenB OS Derivative CONJ-1Glycoconjugate

[0097] The CONJ-1 glycoconjugate was characterized as follows. In orderto demonstrate covalence (e.g., establishing a covalent linkage betweenthe NPr-MenB OS and the protein carrier), a number of physico-chemicaltechniques can be used, including: SDS-PAGE; Western Blot; SephadexG-100 gel filtration; amino acid analysis; or the like. For the purposesof the present study, SDS-PAGE was used to establish covalent attachmentof the NPR-MenB OS/TT CONJ-1 glycoconjugates by revealing a shift tohigher molecular weight for the conjugate band as compared to thecarrier protein band, per se. Western blot analysis of the CONJ-1glycoconjugates demonstrated covalence by the coincidence of positivesignals for TT and NPr-MenB OS with specific anti-TT and anti-NPr-MenBOS antisera.

[0098] Based on steric factors, the use of oligosaccharides instead oflarge molecular weight polysaccharides in the preparation of the CONJ-1glycoconjugates allows for higher coupling efficiency of saccharideantigens onto the protein carrier molecule. The finalsaccharide-to-protein ratio of these NPr-MenB oligosaccharide-basedconjugates range from about 0.10 to 0.25 which corresponds to about 3 to5 NPr-MenB oligosaccharide chains covalently bound per protein carrier.On a per weight basis, the CONJ-1 glycoconjugates appear to have ahigher saccharide loading than a previously reported NPr-MenB PS-basedconjugate (U.S. Pat. No. 4,727,136) which contains, on the average,about 7.5 to 18.8 times more saccharide (using 10,000 Daltons as themolecular weight of NPr-MenB PS).

[0099] In addition, constructing the CONJ-1 glycoconjugates to havesubstantially homogenous-sized saccharide moieties of an intermediatechain length (e.g., average Dp of 10-20) is expected to result inglycoconjugates which display more consistent immunological behavior.Further, the selective end-activation (e.g., selective introduction ofthe aldehyde group at the non-reducing terminus) of the Q-Sepharosechromatography-purified NPr-MenB oligosaccharides avoids the possibilityof cross-linked, heterogenous structures which could arise from the useof NPr-MenB PS molecules with “active” aldehyde groups introduced atboth termini. In this regard, it is likely that bi-terminally activatedpolysaccharide molecules (having aldehyde groups at both ends) could bederived from a periodate oxidation of N-acylated MenB PS previouslyexposed to NaBH₄ during the N-deacetylation procedure.

EXAMPLE 7 Evaluation of Immunogenicity of the NPr-MenB OS DerivativeGlycoconjuqates

[0100] Groups of 4 to 6 week old CD1 and BALB/c mice, 5 to 6 animals pergroup, were vaccinated with 3 doses of a vaccine composition formed fromNPr-MenB OS/TT (CONJ-1) glycoconjugate and FCA adjuvant. A negativecontrol group was vaccinated with the FCA adjuvant alone. Vaccinationsand boosts were administered 3.5 to 4 weeks apart. Pooled sera,collected after the second boost, were analyzed for ELISA titers toNPr-MenB OS. As a positive control, pooled sera from a second post-boostimmunization of CD1 mice vaccinated with a second conjugate, NPr-MenBOS-CRM₁₉₇ (termed CONJ-1/CRM₁₉₇) was used. Antibody specificity was alsodetermined using soluble NPr-MenB OS inhibitor (25 μg/mL) in acompetitive inhibition ELISA. Inhibition by soluble native MenB PS(NAc-MenB PS) (25 μg/mL) was also measured. For the ELISAs, biotinylatedNPr-MenB PS (bound to avidin coated plates) was used as the coatingantigen. The labelling antibody was anti-murine Ig (anti-IgM, IgG andIgA) conjugated to alkaline phosphatase. The results of the ELISAs aredepicted below in Table 3. TABLE 3 % Inhibition¹ MenB Mice Pooled1/Titer Polysaccharides Group Sera Vaccine Adjuvant (OD 1.0) NPr NAc CD1CONJ-1 FCA ˜12,800 90 12 (TT) conjugate BALB/c CONJ-1 FCA ˜3,200 89 4(TT) conjugate Negative NPr-MenB FCA <100 — — control sera OS FCA(CRM₁₉₇) Conjugate Positive NPr-MenB FCA ˜1,600 87 11 control sera* OS(CRM₁₉₇) Conjugate

[0101] The data depicted in Table 3 represent titers obtained using thenet OD after subtraction of OD values in wells containing serum dilutedwith soluble NPr-MenB PS so as to report only inhibitable binding. Thisprocedure avoids reporting of nonspecific binding in the assay (see,e.g., Granoff et al. (1995) Clinic. Diag. Lab. Immunol. 2:574). Percentinhibition with the soluble molecules also is reported as compared withbuffer controls. Antibody response elicited by the CONJ-1glycoconjugates was specific for NPr-MenB saccharide derivatives asevidenced by 80-90% competitive inhibition by the soluble NPr-MenB PS.In contrast, only 4-12% inhibition was observed when soluble NAc-MenB PSwas used. As can be seen, both CD1 and BALB/c mice gave significantantibody responses to NPr-MenB PS when immunized with the CONJ-1glycoconjugates as compared with the negative controls (immunizationswith FCA only). ELISA titers in CD1 mice were higher than those obtainedwith the BALB/c mice.

[0102] Thus, novel MenB OS derivative-immunogens, and methods forobtaining and using the same are disclosed. Although preferredembodiments of the subject invention have been described in some detail,it is understood that obvious variations can be made without departingfrom the spirit and the scope of the invention as defined by theappended claims.

1. A glycoconjugate comprising a Neisseria meningitidis serogroup Bcapsular oligosaccharide (MenB OS) derivative in which sialic acidresidue N-acetyl groups are replaced with N-acyl groups, wherein saidMenB OS derivative is covalently attached to a carrier molecule and hasan average degree of polymerization (Dp) of about 10 to about
 20. 2. Theglycoconjugate of claim 1 wherein the N-acetyl groups are replaced withN-propionyl groups.
 3. The glycoconjugate of claim 1 wherein the carriermolecule is a bacterial toxoid.
 4. The glycoconjugate of claim 3 whereinthe bacterial toxoid is tetanus toxoid.
 5. The glycoconjugate of claim 1wherein the carrier molecule is a nontoxic mutant bacterial toxoid. 6.The glycoconjugate of claim 5 wherein the mutant bacterial toxoid isCRM₁₉₇.
 7. The glycoconjugate of claim 1 wherein the MenB OS derivativehas an average Dp of about 12 to about
 18. 8. A glycoconjugatecomprising a Neisseria meningitidis serogroup B capsular oligosaccharide(MenB OS) derivative in which sialic acid residue N-acetyl groups arereplaced with N-propionyl groups, wherein said MenB OS derivative iscovalently attached to a CRM₁₉₇ toxoid protein carrier and has anaverage Dp of about 12 to about
 18. 9. The glycoconjugate of claim 1wherein the MenB OS derivative further comprises a C3-C16 long-chainaliphatic lipid covalently attached thereto.
 10. The glycoconjugate ofclaim 8 wherein the MenB OS derivative further comprises a C3-C16long-chain aliphatic lipid covalently attached thereto.
 11. A method forproducing a glycoconjugate comprising: (a) providing a heterogenouspopulation of Neisseria meningitidis serogroup B capsularoligosaccharide (MenB OS) derivatives in which sialic acid residueN-acetyl groups are replaced with N-acyl groups; (b) obtaining asubstantially homogenous group of MenB OS derivatives from thepopulation of (a) wherein said group of MenB OS derivatives has anaverage Dp of about 10 to 20; (c) introducing a reactive group at anonreducing end of the derivatives obtained in step (b) to providesingle end-activated MenB OS derivatives; and (d) covalently attachingthe end-activated MenB OS derivatives to a carrier molecule to provide aMenB OS glycoconjugate comprising substantially homogenous sized MenB OSmoieties.
 12. The method of claim 11 wherein the reactive groupintroduced in step (c) comprises a reactive aldehyde group.
 13. Themethod of claim 11 wherein the sialic acid residue N-acetyl groups ofthe MenB OS derivatives are replaced with N-propionyl groups.
 14. Themethod of claim 13 wherein the carrier molecule is a bacterial toxoid.15. The method of claim 13 wherein the carrier molecule is a nontoxicmutant bacterial toxoid.
 16. The method of claim 11 wherein the MenB OSderivative has an average Dp of about 12 to about
 18. 17. The method ofclaim 11 wherein the MenB OS derivative further comprises a C3-C16long-chain aliphatic lipid covalently attached thereto.
 18. A method forproducing a glycoconjugate comprising: (a) providing a heterogenouspopulation of Neisseria meningitidis serogroup B capsularoligosaccharide (MenB OS) derivatives in which sialic acid residueN-acetyl groups are replaced with N-propionyl groups; (b) obtaining asubstantially homogenous group of MenB OS derivatives from thepopulation of (a) wherein said MenB OS derivatives have an average Dp ofabout 12 to 18; (c) introducing a reactive group at a nonreducing end ofthe derivatives obtained in step (b) to provide single end-activatedMenB OS derivatives; and (d) covalently attaching the end-activated MenBOS derivatives to a CRM₁₉₇ bacterial toxoid carrier molecule to providea MenB OS/CRM₁₉₇ toxoid glycoconjugate comprising substantiallyhomogenous sized MenB OS moieties.
 19. The method of claim 18 whereinthe MenB OS derivative further comprises a C3-C16 long-chain aliphaticlipid covalently attached thereto.
 20. A method for producing aglycoconjugate comprising: (a) providing a heterogenous population ofNeisseria meningitidis serogroup B capsular oligosaccharide (MenB OS)derivatives in which sialic acid residue N-acetyl groups are replacedwith N-acyl groups; (b) obtaining a substantially homogenous group ofMenB OS derivatives from the population of (a) wherein said group ofMenB OS derivatives has an average Dp of about 10 to 20; (c) introducinga reactive group at a reducing end of the derivatives obtained in step(b) to provide single end-activated MenB OS derivatives; and (d)covalently attaching the end-activated MenB OS derivatives to a carriermolecule to provide a MenB OS glycoconjugate comprising substantiallyhomogenous sized MenB OS moieties.
 21. The method of claim 20 whereinthe reactive group introduced in step (c) comprises an active estergroup.
 22. The method of claim 20 wherein the sialic acid residueN-acetyl groups of the MenB OS derivatives are replaced with N-propionylgroups.
 23. The method of claim 22 wherein the carrier molecule is abacterial toxoid.
 24. The method of claim 22 wherein the carriermolecule is a nontoxic mutant bacterial toxoid.
 25. The method of claim20 wherein the MenB OS derivative has an average Dp of about 12 to about18.
 26. The method of claim 20 wherein the MenB OS derivative furthercomprises a C3-C16 long-chain aliphatic lipid covalently attachedthereto.
 27. A method for producing a glycoconjugate comprising: (a)providing a heterogenous population of Neisseria meningitidis serogroupB capsular oligosaccharide (MenB OS) derivatives in which sialic acidresidue N-acetyl groups are replaced with N-propionyl groups; (b)obtaining a substantially homogenous group of MenB OS derivatives fromthe population of (a) wherein said MenB OS derivatives have an averageDp of about 12 to 18; (c) introducing a reactive group at a reducing endof the derivatives obtained in step (b) to provide single end-activatedMenB OS derivatives; and (d) covalently attaching the end-activated MenBOS derivatives to a CRM₁₉₇ bacterial toxoid carrier molecule to providea MenB OS/CRM₁₉₇ toxoid glycoconjugate comprising substantiallyhomogenous sized MenB OS moieties.
 28. The method of claim 27 whereinthe MenB OS derivative further comprises a C3-C16 long-chain aliphaticlipid covalently attached thereto.
 29. A glycoconjugate produced by themethod of claim
 11. 30. A glycoconjugate produced by the method of claim18.
 31. A glycoconjugate produced by the method of claim
 20. 32. Aglycoconjugate produced by the method of claim
 27. 33. A vaccinecomposition comprising the combination of: a glycoconjugate formed froma Neisseria meningitidis serogroup B capsular oligosaccharide (MenB OS)derivative in which sialic acid residue N-acetyl groups are replacedwith N-acyl groups, wherein said MenB OS derivative is covalentlyattached to a carrier molecule and has an average degree ofpolymerization (Dp) of about 10 to about 20; and a pharmaceuticallyacceptable excipient.
 34. The vaccine composition of claim 33 whereinthe N-acetyl groups of the MenB OS derivative are replaced withN-propionyl groups.
 35. The vaccine composition of claim 33 wherein theMenB OS derivative has an average Dp of about 12 to about
 18. 36. Thevaccine composition of claim 33 wherein the MenB OS derivative furthercomprises a C3-C16 long-chain aliphatic lipid covalently attachedthereto.
 37. A vaccine composition comprising the combination of: aglycoconjugate formed from a Neisseria meningitidis serogroup B capsularoligosaccharide (MenB OS) derivative in which sialic acid residueN-acetyl groups are replaced with N-propionyl groups, wherein said MenBOS derivative is covalently attached to a CRM₁₉₇ toxoid protein carrierand has an average degree of polymerization (Dp) of about 12 to about18; and a pharmaceutically acceptable excipient.
 38. The vaccinecomposition of claim 37 wherein the MenB OS derivative further comprisesa C3-C16 long-chain aliphatic lipid covalently attached thereto.
 39. Thevaccine composition of claim 33 further comprising an adjuvant.
 40. Thevaccine composition of claim 37 further comprising an adjuvant.
 41. Amethod for preventing Neisseria meningitidis serogroup B and/or E. coliK1 disease in a mammalian subject comprising administering atherapeutically effective amount of the vaccine of claim 33 to saidsubject.
 42. A method for preventing Neisseria meningitidis serogroup Band/or E. coli K1 disease in a mammalian subject comprisingadministering a therapeutically effective amount of the vaccine of claim37 to said subject.